PROCESS FOR PRODUCING v-COELENTERAZINE COMPOUNDS

ABSTRACT

A simple process for producing ν-coelenterazine compounds has been desired. Described is a process for producing a ν-coelenterazine compound represented by general formula (II) comprising (1) the step of reacting a compound of general formula (VIII) with a methyltriphenylphosphonium salt in the presence of a base to give a compound represented by general formula (IX), (2) the step of performing a ring-closing metathesis reaction on any one selected from the group consisting of the compound represented by general formula (IX) and a compound of general formula (X) which is the compound of general formula (IX) wherein the amino is protected with R 5 , and then deprotecting R 4  and, if any, R 5  to give a ν-coelenteramine compound represented by general formula (XIV), and (3) the step of reacting the compound of general formula (XIV) with a compound represented by general formula (XV) to give the compound of general formula (II).

TECHNICAL FIELD

The present invention relates to a process for producing ν-coelenterazine compounds, and so on.

BACKGROUND ART

The development of new drugs is still being actively pursued all over the world, but it is difficult to conclude that new drugs could be efficiently developed as compared to the efforts involved in the development. This is believed to be due to yet insufficient understanding of biomolecular functions. In fact, even the currently available drugs remain yet unclear, in many cases, about how they act upon the body to exhibit their pharmaceutical effects. For this reason, it is required for the development of new drugs to synthesize a number of candidate compounds. Accordingly, if the details of biomolecular mechanism involved in diseases can be clarified, the development of new drugs will be greatly streamlined and such is expected to contribute to the development of a groundbreaking molecular target drug. Recently, trace analysis has become required for the same subject in animal experiments with a new drug. It has thus been strongly desired to develop a noninvasive in vivo imaging technique also from moral and ethical aspects of laboratory animals.

In recent years, as represented by green fluorescence protein (GFP), the imaging technology that visualizes molecules involved in life phenomena has been playing an important role in the medical and biological fields as an innovative technique for elucidating biomolecular functions. On the other hand, techniques for imaging the desired molecules in living animals (in vivo molecular imaging) are still less developed and, above all, it has been actively investigated to develop molecular probes available for fluorescent or luminescent imaging in the near-infrared region (wavelength of 700 nm or longer) which is able to permeate through the living tissues (K. Kiyose et al., Chem. Asian J. 2008, 3, 506).

The present inventors thought that if a practical near-infrared bioluminescence system is newly developed, a breakthrough in vivo imaging technology would be provided and have continued studies, focusing anew on coelenterazine (CTZ, 1) which is a typical bioluminescence substrate from a long time ago.

CTZ is commonly used as a luminescence substrate in many marine organisms, including photoprotein aequorin form jellyfish, Renilla luciferase from sea pansy, Gaussia luciferase from copepoda, Oplophorus luciferase from decapoda, etc. Accordingly, if a near-infrared luminescent CTZ compound (CTZ and its analogues) available for any of luciferases can be developed by modifying the structure of CTZ, it is expected that a luminescence imaging method applicable also to living animals could be provided for the detection with high sensitivity of in vivo localization of a target protein and its absolute quantity and metabolic rate, a promoter or enhancer activity of the target protein, life phenomena of a target cell accompanied by in vivo localization, etc.

Approximately 50 kinds of CTZ compounds have been synthesized so far. Some of them were examined for their substrate specificity in several bioluminescence systems.

Among them, ν-coelenterazine (ν-CTZ, 2) first reported by Shimomura, Kishi, et al. (O. Shimomura, Y. Kishi, et al., Biochem. J. 1988, 251, 405) was later studied for the luminescence properties of aequorin, Renilla luciferase and Oplophorus luciferase by Inouye and Shimomura. As a result, it was found that when used as a substrate for Renilla luciferase, the maximum emission wavelength was shifted toward the longer wavelength side, i.e., from 475 nm (blue emission) to 512 nm (green emission) by about 40 nm (S. Inouye & O. Shimomura, Biochem. Biophys. Res. Commun. 1997, 233, 349). It was also revealed that a part (˜5%) of this emission spectrum distribution reached the near-infrared region (>700 nm).

In addition, A. M. Loenig et al. recently achieved a longer wavelength shift by modifying the amino acid sequence of Renilla luciferase (A. M. Loenig et al., Nature Methods 2007, 4, 641). More specifically, they reported that the maximum emission wavelength was shifted to 547 nm (green emission) when ν-CTZ was used as a substrate for the modified Renilla luciferase. In fact, they also succeeded in imaging in living mice by utilizing this luminescence system.

In order to establish a practical in vivo imaging system, however, it is desired to obtain an emission peak at a longer wavelength region. It is further pointed out that ν-CTZ is easily oxidized and a problem also arises in stability (G. Stepanyuk et al., Anal. Bioanal. Chem. 2010, 398, 1809). It is therefore an important task to create a novel substrate practically applicable to the CTZ near-infrared bioluminescence system.

Furthermore, a process for producing ν-CTZ has not been hitherto reported in detail, and it is also desired to establish the process.

DISCLOSURE OF INVENTION

Under the foregoing circumstances, a simple process for producing ν-coelenterazine compounds has been desired.

In order to solve the problems described above, the present inventors have made extensive investigations and as a result, have succeeded in developing a simple and flexible process for producing ν-coelenterazine, which is applicable also to the production of a variety of ν-coelenterazine analogues.

That is, the present invention provides the process for producing the following ν-coelenterazine compounds, and so on.

[1] A process for producing a ν-coelenteramine compound represented by general formula (XIV):

(wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterocyclic group), which comprises:

(1) the step of reacting a compound represented by general formula (VIII):

(wherein R¹ is the same as defined above and R⁴ is a protecting group) with a methyltriphenylphosphonium salt in the presence of a base to give a compound represented by general formula (IX):

(wherein R¹ and R⁴ are the same as defined above), and,

(2) the step of performing a ring-closing metathesis reaction on any one selected from the group consisting of the compound represented by general formula (IX) and a compound represented by general formula (X) which is the compound represented by general formula (IX) wherein the amino is protected with R⁵:

(wherein R¹ and R⁴ are the same as defined above and R⁵ is a protecting group), and then deprotecting R⁴ and, if any, R⁵.

[2] A process for producing a ν-coelenterazine compound represented by general formula (II):

(wherein:

-   -   R¹ is hydrogen, a halogen, a substituted or unsubstituted         hydrocarbon group, or a substituted or unsubstituted         heterocyclic group,     -   each of R² and R³ independently represents hydrogen, hydroxy, an         alkoxyl, a halogen, or a substituted or unsubstituted         hydrocarbon group), which comprises:

(1) the step of reacting a compound represented by general formula (VIII):

(wherein R¹ is the same as defined above and R⁴ is a protecting group) with a methyltriphenylphosphonium salt in the presence of a base to give a compound represented by general formula (IX):

(wherein R¹ and R⁴ are the same as defined above),

(2) the step of performing a ring-closing metathesis reaction on any one selected from the group consisting of the compound represented by general formula (IX) and a compound represented by general formula (X) which is the compound represented by general formula (IX) wherein the amino is protected with R⁵:

(wherein R¹ and R⁴ are the same as defined above and R⁵ is a protecting group), and then deprotecting R⁴ and if any, R⁵ to give a ν-coelenteramine compound represented by general formula (XIV):

(wherein R¹ is the same as defined above), and,

(3) the step of reacting the compound represented by general formula (XIV) with a compound represented by general formula (XV):

(wherein each of R² and R³ independently represents hydrogen, hydroxy, an alkoxyl, a halogen, a hydrocarbon group, or a hydroxy group protected with a protecting group) to give the compound represented by general formula (II).

[3] The method according to [1] or [2] described above, wherein at least one base selected from the group consisting of n-butyl lithium, potassium tert-butoxide, sodium methoxide, sodium ethoxide and lithium diisopropylamide is used as the base in the step (1) above.

[4] The method according to any one of [1] to [3] described above, wherein at least one solvent selected from the group consisting of tetrahydrofuran, diethyl ether, cyclopropyl methyl ether, tert-butyl methyl ether, dioxane and toluene is used in the step (1) above.

[5] The method according to any one of [1] to [4] above, wherein the reaction temperature and reaction time in the step (1) above are set at 0° C. to 40° C. for an hour to 4 hours.

[6] The method according to any one of [1] to [5] above, wherein a Hoveyda-Grubbs second generation catalyst is used as the catalyst for the ring-closing metathesis reaction in the step (2) above.

[7] The method according to any one of [1] to [6] above, wherein at least one solvent selected from the group consisting of dichloroethane, dichloromethane, chloroform, trichloroethane, tetrachloroethane, benzene, toluene, xylene, monochlorobenzene, dichlorobenzene, hexane, heptane, octane, tetrahydrofuran, dioxane, diethyl ether, dibutyl ether, diisopropyl ether and dimethoxyethane.

[8] The method according to any one of [1] to [7] above, wherein the reaction temperature and reaction time of the ring-closing metathesis reaction in the step (2) above are set at 25° C. to 110° C. for an hour to 48 hours.

[9] The method according to any one of [1] to [8] above, wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted aryl, a substituted or unsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl, an alkyl which may optionally be substituted with an alicyclic group, an alkenyl which may optionally be substituted with an alicyclic group, an alicyclic group, a heterocyclic group, or an alkynyl which may optionally be substituted with an alicyclic group, in the formulae above.

[10] The method according to any one of [1] to [9] above, wherein each of R² and R³ independently represents hydrogen, hydroxy, a halogen, an alkyl having 1 to 4 carbon atoms which may optionally substituted with an alicyclic group, trifluoromethyl or an alkoxyl, in the formula above.

[11] The method according to any one of [1] to [10] above, wherein R⁴ is methyl, methoxymethyl, tetrahydropyranyl, benzyl, 4-methoxybenzyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, in the formulae above.

[12] The method according to any one of [1] to [11] above, wherein R⁵ is acetyl, benzoyl, p-tosyl, tert-butoxycarbonyl or benzyloxycarbonyl, in the formulae above.

[13] The method according to any one of [2] to [12] above, wherein each of the protecting groups for the hydroxy groups for R^(2′) and R^(3′) independently represents tert-butyldimethylsilyl, methoxymethyl, tetrahydropyranyl, trimethylsilyl, phenyldimethylsilyl, triethylsilyl, phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, in the formula above.

[14] The method according to any one of [1] to [13] above, wherein the compound represented by general formula (VIII) above is obtained by:

(1) reacting 2-amino-3,5-dibromo-6-chloropyrazine with R¹MgX (wherein R¹ is the same as defined above and X is a halogen) and ZnCl₂ in the presence of a palladium catalyst to give the compound represented by general formula (V):

(wherein R¹ is the same as defined above),

(2) reacting the compound represented by general formula (V) above with a compound represented by general formula (VI):

(wherein R⁴ is the same as defined above), in the presence of a palladium catalyst and a base to give the compound represented by general formula (VII):

(wherein R¹ and R⁴ are the same as defined above), and,

(3) reacting the compound represented by general formula (VII) with tributyl(vinyl)tin in the presence of a palladium catalyst.

According to the present invention, there is provided a process for producing the ν-coelenterazine compounds in a simple manner. In a preferred embodiment of the present invention, the ν-coelenterazine compounds can be produced in a high yield.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail.

1. ν-Coelenterazine Compound

The present invention provides the compounds represented by general formula (II) below (ν-coelenterazine compounds of the present invention).

In the formula:

-   -   R¹ is hydrogen, a halogen, a substituted or unsubstituted         hydrocarbon group, or a substituted or unsubstituted         heterocyclic group,     -   each of R² and R³ independently represents hydrogen, hydroxy, an         alkoxyl, a halogen, or a substituted or unsubstituted         hydrocarbon group.

In a preferred embodiment of the present invention, the substituted or unsubstituted hydrocarbon group and the substituted or unsubstituted heterocyclic group in R¹ to R³ do not substantially inhibit any of the reactions in the process for producing the ν-coelenterazine compounds or ν-coelenteramine compounds of the present invention.

In a further preferred embodiment of the present invention, in the formulae described above,

-   -   R¹ is hydrogen, a halogen, a substituted or unsubstituted aryl,         a substituted or unsubstituted arylalkyl, a substituted or         unsubstituted arylalkenyl, an alkyl which may optionally be         substituted with an alicyclic group, an alkenyl which may         optionally be substituted with an alicyclic group, an alicyclic         group, a heterocyclic group or an alkynyl which may optionally         be substituted with an alicyclic group, and, each of R² and R³         independently represents hydrogen, hydroxy, a halogen, an alkyl         having 1 to 4 carbon atoms which may optionally substituted with         an alicyclic group, trifluoromethyl or an alkoxyl.

The “halogen” for R¹ includes, for example, fluorine, chlorine, bromine and iodine. In a preferred embodiment of the present invention, the “halogen” is fluorine.

The “substituted or unsubstituted aryl” for R¹ includes, for example, an aryl having 1 to 5 substituents or an unsubstituted aryl. The substituent is at least one selected from the group consisting of a halogen (fluorine, chlorine, bromine, iodine, etc.), hydroxy, an alkyl having 1 to 6 carbon atoms, an alkoxyl having 1 to 6 carbon atoms, an amino, a dialkylamino having 1 to 6 carbon atoms, and the like. In some embodiments of the present invention, the substituent is hydroxy. Specific examples of the “substituted or unsubstituted aryl” include phenyl, p-hydroxyphenyl, p-aminophenyl, p-dimethylaminophenyl, etc. In some embodiments of the present invention, the “substituted or unsubstituted aryl” is an unsubstituted aryl, e.g., phenyl, etc.

The “substituted or unsubstituted arylalkyl” for R¹ includes, for example, an arylalkyl having 7 to 10 carbon atoms which contains 1 to 5 substituents, or an unsubstituted arylalkyl having 7 to 10 carbon atoms. Examples of the substituent are a halogen (fluorine, chlorine, bromine, iodine, etc.), hydroxy, an alkyl having 1 to 6 carbon atoms, an alkoxyl having 1 to 6 carbon atoms, an amino, a dialkylamino having 1 to 6 carbon atoms, and the like. The “substituted or unsubstituted arylalkyl” includes, for example, benzyl, α-hydroxybenzyl, phenylethyl, p-hydroxybenzyl, p-dimethylaminobenzyl, etc., preferably, benzyl, α-hydroxybenzyl, phenylethyl, etc. In some embodiments of the present invention, the “substituted or unsubstituted arylalkyl” is benzyl.

The “substituted or unsubstituted arylalkenyl” for R¹ includes, for example, an arylalkenyl having 8 to 10 carbon atoms which contain 1 to 5 substituents, or an unsubstituted arylalkenyl having 8 to 10 carbon atoms. Examples of the substituent are a halogen (fluorine, chlorine, bromine, iodine, etc.), hydroxy, an alkyl having 1 to 6 carbon atoms, an alkoxyl having 1 to 6 carbon atoms, an amino, a dialkylamino having 1 to 6 carbon atoms, and the like. Examples of the “substituted or unsubstituted arylalkenyl” include phenylvinyl, p-hydroxyphenylvinyl, p-dimethylaminophenylvinyl, etc. In some embodiments of the present invention, the “substituted or unsubstituted arylalkenyl” is an unsubstituted arylalkenyl, e.g., phenylvinyl, etc.

The “alkyl which may optionally be substituted with an alicyclic group” for R¹ is, for example, an unsubstituted straight or branched alkyl having 1 to 4 carbon atoms, or an unsubstituted straight or branched alkyl having 1 to 4 carbon atoms which is substituted with, e.g., 1 to 10 alicyclic groups. Examples of the alicyclic group are cyclohexyl, cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples of the “alkyl which may optionally be substituted with an alicyclic group” are methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentyl methyl, cyclohexylmethyl, cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl, etc., preferably, methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc. In some embodiments of the present invention, the “alkyl which may optionally be substituted with an alicyclic group” is a straight alkyl which may optionally be substituted with an alicyclic group, and examples include methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc.

The “alkenyl which may optionally be substituted with an alicyclic group” for R¹ is an unsubstituted straight or branched alkenyl having 2 to 6 carbon atoms, or a straight or branched alkenyl having 2 to 6 carbon atoms which is substituted with, e.g., 1 to 10 alicyclic groups. Examples of the alicyclic group are cyclohexyl, cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples of the “alkenyl which may optionally be substituted with an alicyclic group” include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylpropenyl, etc., preferably, 2-methylpropenyl, etc.

Examples of the “alicyclic group” for R¹ include cyclohexyl, cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl, etc.

The “heterocyclic group” for R¹ is, for example, a group formed by a 5- to 7-membered ring containing 1 to 3 atoms selected from the group consisting of N, O and S as the atoms for forming the ring, in addition to carbons and bound through carbon, a group formed by condensing at least two such rings and bound through carbon, or a group formed by condensing such rings with a benzene ring and bound through carbon. Examples of the “heterocyclic group” are thiophen-2-yl, 2-furanyl, 4-pyridyl, etc. In some embodiments of the present invention, the “heterocyclic group” is a heterocyclic group containing sulfur, e.g., thiophen-2-yl.

The “alkynyl which may optionally be substituted with an alicyclic group” for R¹ is an unsubstituted straight or branched alkynyl having 2 to 6 carbon atoms, or a substituted straight or branched alkynyl having 2 to 6 carbon atoms which is optionally substituted with, e.g., 1 to 10 alicyclic groups. Examples of the alicyclic group are cyclohexyl, cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples of the “alkynyl which may optionally be substituted with an alicyclic group” are ethynyl, propynyl, butynyl, 2-methylpropynyl, etc., preferably, 2-methylpropynyl, etc.

In a preferred embodiment of the present invention, R¹ is phenyl, p-hydroxyphenyl, benzyl, α-hydroxybenzyl, phenylethyl, phenylvinyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl, methyl, ethyl, propyl, 2-methylpropyl, 2-methylpropenyl, adamantylmethyl, cyclopentylmethyl or thiophen-2-yl. In a more preferred embodiment of the present invention, R¹ is benzyl.

The “halogen” for R² includes, for example, fluorine, chlorine, bromine and iodine. In a preferred embodiment of the present invention, the “halogen” is fluorine.

The “alkyl having 1 to 4 carbon atoms which may optionally be substituted with an aliphatic group” for R² is, for example, an unsubstituted straight or branched alkyl having 1 to 4 carbon atoms or a straight or branched alkyl having 1 to 4 carbon atoms which is substituted with, e.g., 1 to 10 alicyclic groups. Examples of the alicyclic group are cyclohexyl, cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples of the “alkyl which may optionally be substituted with an alicyclic group” are methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexyl methyl, cyclohexylethyl, cyclobutylmethyl, cyclopropylmethyl, etc., preferably, methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc. In some embodiments of the present invention, the “alkyl which may optionally be substituted with an alicyclic group” is a straight alkyl which may optionally be substituted with an alicyclic group, and examples include methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl, cyclohexyl methyl, cyclohexylethyl, etc.

The “alkoxyl” for R² includes, for example, a straight or branched alkoxy having 1 to 6 carbon atoms. Examples of the “alkoxy” are methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy, iso-pentyloxy, sec-pentyloxy, 1,1-dimethylpropyloxy, 1,2-dimethylpropoxy, 2,2-dimethylpropyloxy, n-hexoxy, 1-ethylpropoxy, 2-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, iso-hexoxy, 1-methyl-2-ethylpropoxy, 1-ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1,2-trimethylpropoxy, 1-propylpropoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy, 2-ethylbutoxy, 1,3-dimethylbutoxy, 2-methylpentoxy, 3-methylpentoxy, hexyloxy, etc. In some embodiments of the present invention, the “alkoxy” is methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, etc., preferably, methoxy.

In a preferred embodiment of the present invention, R² is hydrogen, hydroxy, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, trifluoromethyl, methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy or tert-butoxy. In a more preferred embodiment of the present invention, R² is hydroxy or trifluoromethyl.

The “halogen” for R³ includes, for example, fluorine, chlorine, bromine and iodine. In a preferred embodiment of the present invention, the “halogen” is fluorine.

The “alkyl having 1 to 4 carbon atoms which may optionally be substituted with an alicyclic group” for R³ includes, for example, an unsubstituted straight or branched alkyl having 1 to 4 carbon atoms or a substituted straight or branched alkyl having 1 to 4 carbon atoms which is substituted with, e.g., 1 to 10 alicyclic groups. Examples of the alicyclic group are cyclohexyl, cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl, cyclopentyl, adamantyl, etc. Examples of the “alkyl which may optionally be substituted with an alicyclic group” are methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, cyclobutyl methyl, cyclopropylmethyl, etc., preferably, methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc. In some embodiments of the present invention, the “alkyl which may optionally be substituted with an alicyclic group” is a straight alkyl which may optionally be substituted with an alicyclic group, and examples include methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, etc.

The “alkoxyl” for R³ includes, for example, a straight or branched “alkoxy” having 1 to 6 carbon atoms and examples of the “alkoxy” are methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy, iso-pentyloxy, sec-pentyloxy, 1,1-dimethylpropyloxy, 1,2-dimethylpropoxy, 2,2-dimethylpropyloxy, n-hexoxy, 1-ethylpropoxy, 2-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, iso-hexoxy, 1-methyl-2-ethylpropoxy, 1-ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1,2-trimethylpropoxy, 1-propylpropoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy, 2-ethylbutoxy, 1,3-dimethylbutoxy, 2-methylpentoxy, 3-methylpentoxy, hexyloxy, etc. In some embodiments of the present invention, the “alkoxy” includes methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and preferably, methoxy.

In a preferred embodiment of the present invention, R³ is hydrogen, hydroxy, fluorine, methyl, ethyl, propyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, trifluoromethyl, methoxy, ethoxy, n-propoxy, iso-propoxy, sec-propoxy, n-butoxy, iso-butoxy, sec-butoxy or tert-butoxy. In a more preferred embodiment of the present invention, R³ is hydrogen.

In some embodiments of the present invention, in the general formula (II):

-   -   R¹ is benzyl,     -   R² is hydroxy or trifluoromethyl, and,     -   R³ is hydrogen.

In a certain embodiment of the present invention, the compound represented by general formula (II) is the compound represented by the formula described below (ν-coelenterazine).

In another embodiment of the present invention, the compound represented by general formula (II) is the compound represented by the following formula (cf3-ν-coelenterazine).

The present invention further provides the compounds represented by general formula (XIV) below (the ν-coelenteramine compounds of the present invention):

(wherein R¹ is the same as described for the general formula (II) above).

In a certain embodiment of the present invention, the compound represented by general formula (XIV) is the compounds represented by the following formula (ν-coelenteramine).

2. Process for Producing ν-Coelenteramine Compounds or ν-Coelenterazine Compounds of the Invention

The process for producing the compound represented by general formula (XIV) (process for producing the ν-coelenteramine compounds of the present invention) and the process for producing the compound represented by general formula (II) (process for producing the ν-coelenterazine compounds of the present invention) are described below.

In the production processes described below, R¹ to R³ are the same as described above.

Each of R^(2′) and R^(3′) independently represents hydrogen, hydroxy, an alkoxyl, a halogen, a substituted or unsubstituted hydrocarbon group or a protected hydroxy, etc. The protecting group in the “hydroxy protected with a protecting group” for R^(2′) and R^(3′) is, for example, tert-butyldimethylsilyl, methoxymethyl, tetrahydropyranyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, preferably, tert-butyldimethylsilyl.

The “alkoxyl,” “halogen” and “substituted or unsubstituted hydrocarbon group” for R^(2′) and R^(3′) are the same as described for R² and R³.

Preferably, R^(2′) is tert-butyldimethylsilyloxy. Preferably, R^(3′) is hydrogen.

R⁴ is a protecting group, e.g., methyl, methoxymethyl, tetrahydropyranyl, benzyl, 4-methoxybenzyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl, tert-butyldiphenylsilyl or triisopropylsilyl, and preferably, methyl.

R⁵ is a protecting group, e.g., acetyl, benzoyl, p-tosyl, tert-butoycarbonyl or benzyloxycarbonyl. Preferably, R⁵ is acetyl.

X is a halogen, e.g., fluorine (F⁻), chlorine (Cl⁻), bromine (Br⁻) or iodine (I⁻). Preferably, X is bromine (Br⁻).

2.1. Process for Producing the Compound Represented by General Formula (XIV)

The compound represented by general formula (XIV) can be produced from the compound represented by general formula (VIII) as follows.

(1) In the first step, the formyl in the compound represented by general formula (VIII) is methylated via a Wittig reaction. More specifically, the compound represented by general formula (VIII) is reacted with a methyltriphenylphosphonium salt (Ph₃P′ CH₃X) in the presence of a base to give the compound represented by general formula (IX).

The methyltriphenylphosphonium salt is preferably methyltriphenylphosphonium bromide.

Specific examples of the base used in the Wittig reaction are n-butyl lithium (n-BuLi), potassium tert-butoxide, sodium methoxide, sodium ethoxide, lithium diisopropylamide, etc.

Specific examples of the solvents used in the reaction include tetrahydrofuran, diethyl ether, cyclopropyl methyl ether, tert-butyl methyl ether, dioxane, toluene, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 0° C. to 40° C. for an hour to 4 hours, preferably, at 10° C. to 30° C. for an hour to 3 hours, and more preferably, at 10° C. to 20° C. for an hour to 2 hours.

(2) In the next step, any one selected from the group consisting of the compound represented by general formula (IX) and the compound represented by general formula (X) in which the amino in the compound represented by general formula (IX) is protected with R⁵ is subjected to the ring-closing metathesis reaction followed by deprotecting R⁴ and, if any, R⁵. Thus, the compound represented by general formula (XIV) is obtained.

(2-1) When the compound represented by general formula (X) is subjected to the subsequent ring-closing metathesis reaction, the amino in the compound represented by general formula (IX) is first protected with R⁵ to give the compound represented by general formula (X).

Specifically, protection of the amino with R⁵ is performed as follows. The compound represented by general formula (IX) is reacted with acetyl chloride, acetic anhydride, benzoyl chloride, di-t-butyl dicarbonate, benzyl chloroformate, etc. in the presence of a base such as triethylamine, diisopropylethylamine, pyridine and 4-dimethylaminopyridine. More specifically, the compound represented by general formula (X) wherein R⁵ is acetyl can be obtained by the process described in SYNTHESIS EXAMPLES later described.

Specific examples of the solvents used in the reaction include pyridine, methylene chloride, tetrahydrofuran, toluene, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 0° C. to 100° C. for an hour to 24 hours, preferably, at 20° C. to 70° C. for an hour to 12 hours, and more preferably, at 50° C. to 60° C. for an hour to 2 hours.

(2-2) Subsequently, any one selected from the group consisting of the compound represented by general formula (IX) and the compound represented by general formula (X) is subjected to the ring-closing metathesis reaction to give a tricyclic aromatic compound. More specifically, the ring-closing metathesis reaction of the compound represented by general formula (IX) or the compound represented by general formula (X) is carried out in the presence of a Grubbs catalyst or a Hoveyda-Grubbs catalyst to give the compound represented by general formula (XI′) or the compound represented by general formula (XI).

In a preferred embodiment of the present invention, the compound represented by general formula (X) is used as the compound provided for the ring-closing metathesis. By using the compound represented by general formula (X), the compound represented by general formula (XI) can be obtained in a good yield.

The Grubbs catalyst or the Hoveyda-Grubbs catalyst used in the ring-closing metathesis reaction includes a Grubbs first generation catalyst, a Grubbs second generation catalyst, a Hoveyda-Grubbs first generation catalyst, a Hoveyda-Grubbs second generation catalyst, etc., and preferably, a Hoveyda-Grubbs second generation catalyst.

Specific examples of the solvents used in the reaction include dichloroethane, dichloromethane, chloroform, trichloroethane, tetrachloroethane, benzene, toluene, xylene, monochlorobenzene, dichlorobenzene, hexane, heptane, octane, tetrahydrofuran, dioxane, diethyl ether, dibutyl ether, diisopropyl ether, dimethoxyethane, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 25° C. to 110° C. for an hour to 48 hours, preferably, at 50° C. to 110° C. for an hour to 24 hours, and more preferably, at 90° C. to 100° C. for 12 to 18 hours.

(2-3) After the compound represented by general formula (XI′) is obtained in (2-2) above, R⁴ in the compound represented by general formula (XI′) is deprotected to give the compound represented by general formula (XIV).

Specifically, R⁴ is deprotected as follows. The deprotection is effected through the reaction with a Lewis acid or a Brønsted acid. More specifically, the deprotection of R⁴ which is methyl can be performed by the procedures described in SYNTHESIS EXAMPLES later given.

Specific examples of the solvents used in the reaction include pyridine, tetrahydrofuran, toluene, dimethylformamide, dimethylsulfoxide, water, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof, or may not be used.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 0° C. to 300° C. for 0.1 to 60 hours, preferably, at 30° C. to 250° C. for 0.5 to 12 hours, and more preferably, at 180° C. to 220° C. for 0.5 to an hour.

(2-4) On the other hand, when the compound represented by general formula (XI) is obtained in (2-2) above, R⁴ in the compound represented by general formula (XI) is deprotected to give the compound represented by general formula (XIII)

Specifically, R⁴ is deprotected as follows. The deprotection is effected by the reaction with a Lewis acid or a Brønsted acid. More specifically, the deprotection of R⁴ which is methyl can be performed by the procedures described in SYNTHESIS EXAMPLES later given.

Specific examples of the solvents used in the reaction include dichloromethane, tetrahydrofuran, diethyl ether, dioxane, methanol, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 0° C. to 50° C. for an hour to 24 hours, preferably, at 0° C. to 30° C. for 2 to 6 hours, and more preferably, at 0° C. to 20° C. for 2 to 4 hours.

(2-5) Furthermore, R⁵ in the compound represented by general formula (XIII) is deprotected to give the compound represented by general formula (XIV).

Specifically, R⁵ is deprotected as follows. The deprotection is effected by heating in a solvent in the presence of a base such as sodium hydrogencarbonate, potassium carbonate, etc. More specifically, the deprotection of R⁵ which is acetyl can be performed by the procedures described in SYNTHESIS EXAMPLES later given.

Specific examples of the solvents used in the reaction include methanol, ethanol, n-butanol, t-butanol, dioxane, tetrahydrofuran, water, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 40° C. to 100° C. for an hour to 24 hours, preferably, at 50° C. to 80° C. for 2 to 24 hours, and more preferably, at 60° C. to 70° C. for 12 to 16 hours.

2.2. Process for Producing the Compound Represented by General Formula (II)

The compound represented by general formula (II) can be produced from the compound represented by general formula (VIII) as follows.

(1) The compound represented by general formula (XIV) is produced as described above.

(2) Then, the compound represented by general formula (XIV) is reacted with the compound represented by general formula (XV) to give the compound represented by general formula (II).

The compound represented by general formula (XV) can be produced by known processes. Specifically, the compound can be produced by the processes described in Adamczyk, M. et al., Synth. Commun., 32, 3199-3205 (2002) or Baganz, H. & May, H.-J. Chem. Ber., 99, 3766-3770 (1966) and Baganz, H. & May, H.-J. Angew. Chem., Int. Ed. Eng., 5, 420 (1966), or modifications thereof. More specifically, the compound represented by general formula (XV) can be produced by reacting a substituted benzyl Grignard reagent with ethyl diethoxyacetate at a low temperature (−78° C.) or by reacting an α-diazo-α′-substituted phenyl ketone with tert-butyl hypochlorite in ethanol.

The solvent used in the reaction to obtain the compound represented by general formula (II) is not particularly limited but various solvent can be used. Examples are dioxane, tetrahydrofuran, ether, methanol, ethanol, water, etc. They may be used alone or as an admixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 0° C. to 200° C. for an hour to 96 hours, at room temperature to 150° C. for 3 to 72 hours, or at 60° C. to 120° C. for 6 to 24 hours.

2.3. Process for Producing the Compound Represented by General Formula (VIII)

The compound represented by general formula (VIII) above can be produced from, e.g., the compound represented by general formula (IV), as follows.

(1) In the first step, the Negishi coupling is performed on the compound represented by general formula (IV) with an organic zinc compound. Specifically, the compound represented by general formula (IV) is reacted with R¹MgX and ZnCl₂ in the presence of a palladium catalyst to give the compound represented by general formula (V).

The compound represented by general formula (IV) is the compound described in WO 2003/059893 and may be synthesized, e.g., by the procedures described in SYNTHESIS EXAMPLES later given.

R¹MgCl used may be synthesized by known synthesis methods or may be commercially available.

Specific examples of the palladium catalyst used for the Negishi coupling include Pd(PPh₃)₄, PdCl₂(PPh₃)₂, Pd(OAc)₂, tris(dibenzylideneacetone)dipalladium(0), tris(dibenzylideneacetone)dipalladium(0) chloroform complex, bis(dibenzylideneacetone)palladium(0), bis(tri-t-butylphosphino)palladium(0), (1,1′-bis(diphenylphosphino)ferrocene)dichloropalladium(II), etc.

Specific examples of the solvents used in the reaction are benzene, toluene, xylene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether, t-butyl methyl ether, 1,4-dioxane, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 0° C. to 50° C. for an hour to 24 hours, preferably, at 10° C. to 40° C. for 2 to 24 hours, and more preferably, at 20° C. to 30° C. for 6 to 16 hours.

(2) In the next step, the compound represented by general formula (V) is arylated at the 5-position selectively via the Suzuki-Miyaura coupling with boronic acid. Specifically, the compound represented by general formula (V) is reacted with the compound represented by general formula (VI) in the presence of a palladium catalyst and a base to give the compound represented by general formula (VII).

The compound represented by general formula (VI) used may be commercially available or may be synthesized by known synthesis methods.

Specific examples of the palladium catalyst used in the Suzuki-Miyaura coupling are allylpalladium(II) chloride (dimer), Pd(PPh₃)₄, PdCl₂(PPh₃)₂, Pd(OAc)₂, tris(dibenzylideneacetone)dipalladium(0), tris(dibenzylideneacetone)dipalladium(0) chloroform complex, bis(dibenzylideneacetone)palladium(0), etc.

Phosphine compounds may be added to these palladium catalyst, if necessary, to accelerate the reaction. Specific examples of the phosphine compounds are

-   2-(di-t-butylphosphino)-1-phenylindole, tri(t-butyl)phosphine,     tricyclohexylphosphine, -   1-(N,N-dimethylaminomethyl)-2-(di-t-butylphosphino)ferrocene, -   1-(N,N-dibutylaminomethyl)-2-(di-t-butylphosphino)ferrocene, -   1-(methoxymethyl)-2-(di-t-butylphosphino)ferrocene, -   1,1′-bis(di-t-butylphosphino)ferrocene, 2,     2′-bis(di-t-butylphosphino)-1,1′-binaphthyl, -   2-methoxy-2′-(di-t-butylphosphino)-1,1′-binaphthyl, -   2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl, etc.

Specific examples of the base used in the reaction include sodium carbonate, potassium carbonate, cesium carbonate, sodium hydrogencarbonate, sodium hydroxide, potassium hydroxide, barium hydroxide, sodium ethoxide, sodium t-butoxide, sodium acetate, tripotassium phosphate, potassium fluoride, etc.

Specific examples of the solvents used in the reaction are benzene, toluene, xylene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether, t-butyl methyl ether, 1,4-dioxane, methanol, ethanol, isopropyl alcohol, water, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 0° C. to 50° C. for an hour to 24 hours, preferably, at 10° C. to 40° C. for 2 to 24 hours, and more preferably, at 20° C. to 30° C. for 6 to 16 hours.

(3) In the next step, the 6-position of the pyrazine ring in the compound represented by general formula (VII) is vinylated through the Stille coupling with a vinyl compound. Specifically, the compound represented by general formula (VII) is reacted with tributyl(vinyl)tin in the presence of a palladium catalyst to give the compound represented by general formula (VIII).

Tributyl(vinyl)tin used may be commercially available or may be synthesized by known methods.

Specific examples of the palladium catalyst used in the Stille coupling are Pd(PPh₃)₄, PdCl₂(PPh₃)₂, Pd(OAc)₂, tris(dibenzylideneacetone)dipalladium(0), tris(dibenzylideneacetone)dipalladium(0) chloroform complex, bis(dibenzylideneacetone)palladium(0), etc.

In order to accelerate the reaction, these palladium catalysts may be added with tetra-n-butylammonium chloride, tetra-n-butylammonium bromide, tetra-n-butylammonium iodide, etc., if necessary. Furthermore, polymerization inhibitors such as 2,6-di-tert-butyl-4-methylphenol (BHT), etc. may also be added to inhibit the formation of by-products.

Specific examples of the solvents used in the reaction are benzene, toluene, xylene, N,N-dimethylformamide, tetrahydrofuran, diethyl ether, t-butyl methyl ether, 1,4-dioxane, etc. These solvents may be appropriately chosen and used alone or as a solvent mixture thereof.

The reaction temperature and reaction time are not particularly limited and are, e.g., at 60° C. to 120° C. for an hour to 24 hours, preferably, at 80° C. to 110° C. for an hour to 12 hours, and more preferably, at 100° C. to 110° C. for an hour to 2 hours.

3. Method for Producing the Calcium-Binding Photoprotein

The calcium-binding photoprotein of the present invention can be produced or regenerated by contacting the compound represented by general formula (II) (the ν-coelenterazine compound of the present invention) with an apoprotein of the calcium-binding photoprotein to give the calcium-binding photoprotein.

As used herein, the term “contact” means that the ν-coelenterazine compound and an apoprotein of the calcium-binding photoprotein are allowed to be present in the same reaction system, and includes, for example, the states that an apoprotein of the calcium-binding photoprotein is added to a container charged with the ν-coelenterazine compound, the ν-coelenterazine compound is added to a container charged with an apoprotein of the calcium-binding photoprotein, the ν-coelenterazine compound is mixed with an apoprotein of the calcium-binding photoprotein, and the like. In one embodiment of the present invention, the contact is effected at a low temperature in the presence of a reducing agent (e.g., mercaptoethanol, dithiothreitol, etc.) and oxygen. More specifically, the photoprotein of the present invention can be produced or regenerated by the methods described in, e.g., Shimomura, O. et al., Biochem. J., 251, 405-410 (1988), Shimomura, O. et al, Biochem. J., 261, 913-920(1989), etc. The calcium-binding photoprotein of the present invention exists in the state that a peroxide of the ν-coelenterazine compound produced from the ν-coelenterazine compound and molecular oxygen and the apoprotein form a complex in the presence of oxygen. When calcium ions bind to the complex described above, the complex emits light to generate the ν-coelenteramide compound as the oxide of the ν-coelenterazine compound and carbon dioxide. The complex described above is sometimes referred to as the “photoprotein of the present invention.”

Examples of the apoprotein used to produce the photoprotein of the present invention include apoaequorin, apoclytin-I, apoclytin-II, apobelin, apomitrocomin, apomineopsin, apobervoin, and the like. In some embodiments of the present invention, the apoprotein is apoaequorin, apobelin, apoclytin-I, apoclytin-II, mitrocomin, etc., e.g., apoaequorin. These apoproteins may be obtained from natural sources or genetically engineered. Furthermore, the amino acid sequence may also be mutated from the native sequence by gene recombination technology, as long as the apoproteins are capable of producing the calcium-binding photoprotein.

The nucleotide sequences and amino acid sequences of the apoproteins of photoproteins obtained from the nature (native apoproteins) are as follows. The nucleotide sequence and amino acid sequence of native apoaequorin are shown by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The nucleotide sequence and amino acid sequence of native apoclytin-I are shown by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The nucleotide sequence and amino acid sequence of native apoclytin-II are shown by SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The nucleotide sequence and amino acid sequence of native apomitrocomin are shown by SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The nucleotide sequence and amino acid sequence of native apobelin are shown by SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The nucleotide sequence and amino acid sequence of native apobervoin are shown by SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

The apoprotein mutated by recombinant technology is, for example, a protein selected from the group consisting of (a) to (c) below:

-   -   (a) a protein comprising the amino acid sequence of native         apoprotein in which 1 or more amino acids are deleted,         substituted, inserted and/or added, and having the activity or         function of the apoprotein of the calcium-binding photoprotein;     -   (b) a protein comprising an amino acid sequence which has 90% or         more homology to the amino acid sequence of native apoprotein,         and having the activity or function of the apoprotein of the         calcium-binding photoprotein; and,     -   (c) a protein comprising an amino acid sequence encoded by a         polynucleotide that hybridizes under stringent conditions to a         polynucleotide comprising a nucleotide sequence complementary to         the nucleotide sequence of native apoprotein, and having the         activity or function of the apoprotein of the calcium-binding         photoprotein.

Examples of the “native apoprotein” described above are apoaequorin, apoclytin-I, apoclytin-II, apobelin, apomitrocomin, apomineopsin, apobervoin, etc. In an embodiment of the present invention, the apoprotein is apoaequorin, apobelin, apoclytin-I, apoclytin-II, apobelin, apomitrocomin, etc., preferably apoaequorin. The amino acid sequences and nucleotide sequences of these native apoproteins are the same as described above.

The term “activity or function of the apoprotein of the calcium-binding photoprotein” described above is used to mean, for example, the activity or function of the apoprotein which binds to a peroxide of the ν-coelenterazine compound to produce the calcium-binding photoprotein. “The protein binds to a peroxide of the ν-coelenterazine compound to produce the calcium-binding photoprotein” specifically means not only (1) that the protein binds to the peroxide of ν-coelenterazine compound to produce the photoprotein, but also (2) that the protein is brought in contact with the ν-coelenterazine compound in the presence of oxygen to produce the photoprotein (complex) containing the protein and a peroxide of the ν-coelenterazine compound. As used herein, the term “contact” means that the protein and the ν-coelenterazine compound are allowed to be present in the same reaction system, and includes, for example, addition of the protein to a container charged with the ν-coelenterazine compound, addition of the ν-coelenterazine compound to a container charged with the protein, mixing of the protein with the ν-coelenterazine compound, and the like.

The “ν-coelenterazine compound” refers to ν-coelenterazine and as in ν-coelenterazine, a compound capable of constituting as the apoprotein the calcium-binding photoprotein such as aequorin, etc. (ν-coelenterazine analogue), specifically, cf3-ν-coelenterazine.

The range of “1 or more” in “the amino acid sequence in which 1 or more amino acids are deleted, substituted, inserted and/or added” described above is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1. In general, the less the number of the amino acids deleted, substituted, inserted or added, the more preferable. In the deletion, substitution, insertion and addition of the amino acid residues described above, two or more may occur concurrently. Such regions can be acquired by using site-directed mutagenesis described in “Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001),” “Ausbel F. M. et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997),” “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982),” “Gene, 34, 315 (1985),” “Nuc. Acids. Res., 13, 4431 (1985),” “Proc. Natl. Acad. Sci. USA, 82, 488 (1985),” etc.

The range of “90% or more” in the “amino acid sequence which has 90% or more homology” is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more. It is generally preferred for the numerical value indicating the degree of homology to be higher. The homology between amino acid sequences or nucleotide sequences can be determined using a sequencing program such as BLAST (see, e.g., Altzshul S. F. et al., J. Mol. Biol., 215, 403 (1990), etc.) or the like. When BLAST is used, the default parameters for the respective programs are used.

The “polynucleotide that hybridizes under stringent conditions” described above refers to a polynucleotide (e.g., DNA) which is obtained by, for example, colony hybridization, plaque hybridization, Southern hybridization, etc., using as a probe all or part of a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of native apoprotein or a polynucleotide encoding the amino acid sequence of native apoprotein. Specific examples include a polynucleotide which can be identified by performing hybridization at 65° C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on which the polynucleotide from a colony or plaque is immobilized, then washing the filter under 65° C. conditions with an SSC (saline-sodium citrate) solution having a concentration of 0.1 to 2 times (a 1×SSC solution is composed of 150 mmol/L of sodium chloride and 15 mmol/L of sodium citrate).

Hybridization may be performed in accordance with modifications of the methods described in textbooks of experiment, e.g., Sambrook J. et al, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel F. M. et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997), Glover D. M. and Hames B. D., DNA Cloning 1: Core Techniques, A practical Approach, Second Edition, Oxford University Press (1995), etc.

As used herein, the term “stringent conditions” may refer to less stringent conditions, moderately stringent conditions and highly stringent conditions. The “less stringent conditions” are, for example, the conditions under 5×SSC, 5× Denhardt's solution, 0.5% (w/v) SDS and 50% (v/v) formamide at 32° C. The “moderately stringent conditions” are, for example, the conditions under 5×SSC, 5× Denhardt's solution, 0.5% (w/v) SDS and 50% (v/v) formamide at 42° C. The “highly stringent conditions” are, for example, the conditions under 5×SSC, 5× Denhardt's solution, 0.5% (w/v) SDS and 50% (v/v) formamide at 50° C. The more stringent the conditions are, the higher the complementarity required for double strand formation. Specifically, for example, under these conditions, a polynucleotide (e.g., DNA) of higher homology is expected to be obtained efficiently at higher temperatures, although multiple factors are involved in hybridization stringency, including temperature, probe concentration, probe length, ionic strength, time and base concentration; those skilled in the art may appropriately select these factors to realize a similar stringency.

When a commercially available kit is used for the hybridization, for example, AlkPhos Direct Labeling Reagents (manufactured by Amersham Pharmacia) can be used. According to the protocol attached to the kit in this case, incubation with a labeled probe is performed overnight, the membrane is then washed with a primary wash buffer containing 0.1% (w/v) SDS at 55° C., and finally the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by a sequencing program such as BLAST or the like using the default parameters, DNAs having homology of approximately 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more, to the polynucleotide encoding the amino acid sequence of the apoprotein. The homology of an amino acid sequence or a nucleotide sequence can be determined using the method described hereinabove.

Examples of the recombinant apoprotein which can be used in the present invention include recombinant aequorin described in Shimomura, O. and Inouye, S. Protein Express. Purif. (1999) 16: 91-95, recombinant clytin-I described in Inouye, S. and Sahara, Y. Protein Express. Purif (2007) 53: 384-389, recombinant clytin-II described in Inouye, S. J. Biochem. (2008) 143: 711-717, and the like.

The calcium-binding photoprotein thus produced may be further purified. Purification of the calcium-binding photoprotein can be performed in a conventional manner of separation/purification. The separation/purification includes, for example, precipitation with ammonium sulfate, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reversed phase high performance liquid chromatography, dialysis, ultrafiltration, etc., alone or in an appropriate combination of these techniques.

4. Use of ν-Coelenterazine Compound of the Invention or Photoprotein of the Invention (1) Use as Luminescence Substrate

The ν-coelenterazine compound in some embodiments of the present invention emits light by the action of an emission-catalyzing enzyme and can be used as a luminescence substrate. Therefore, the present invention provides a method for emitting light which comprises contacting the ν-coelenterazine compound of the present invention with an emission-catalyzing enzyme. As used herein, the term “contact” means that the ν-coelenterazine compound of the present invention and an emission-catalyzing enzyme are allowed to be present in the same reaction system, and includes, for example, the states that the emission-catalyzing enzyme is added to a container charged with the ν-coelenterazine compound, the ν-coelenterazine compound is added to a container charged with the emission-catalyzing enzyme, the ν-coelenterazine compound is mixed with the emission-catalyzing enzyme, and the like.

The emission-catalyzing enzyme used in the method of the present invention for emitting light is, for example, a luciferase derived from Oplophorus sp., e.g., Oplophorus gracilorostris (Oplophorus luciferase), a luciferase derived from Gaussia sp., e.g., Gaussia princeps (Gaussia luciferase), a luciferase derived from Renilla sp., e.g., Renilla reniformis, or Renilla muelleri (Renilla luciferase), a luciferase derived from Pleuromamma sp. (Pleuromamma luciferase) or a luciferase derived from Metridia longa (Metridia luciferase).

The emission-catalyzing enzyme used for the method of the present invention for emitting light may be mutants of these luciferases. Examples of the mutants include mutants of Renilla luciferase described in Loening et al. Protein Eng. Des. Sel. (2006) 19, 391-400, mutants of Renilla luciferase described in Loening et al. Nature methods (2007) 4, 641-643, mutants of Renilla luciferase described in Woo et al. Protein Sci. (2008) 17, 725-735, etc.

These emission-catalyzing enzymes can be prepared by the method described in, e.g., Shimomura et al. (1988) Biochem. J. 251, 405-410, Shimomura et al. (1989) Biochem. J. 261, 913-920, or Shimomura et al. (1990) Biochem. J. 270, 309-312, or modifications thereof. Alternatively, various types of the enzymes are commercially available from JNC Corporation (former Chisso Corporation), Wako Pure Chemical, Promega Inc., etc. and these enzymes commercially available may also be employed for the method of the present invention for emitting light.

Among the Renilla luciferase, the nucleotide sequence and amino acid sequence of Renilla reniformis-derived luciferase are shown by SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The nucleotide sequence and amino acid sequence of Oplophorus gracilorostris-derived luciferase among the Oplophorus luciferase are shown by SEQ ID NO: 15 and SEQ ID NO: 16, respectively. The nucleotide sequence and amino acid sequence of Gaussia princeps-derived luciferase in the Gaussia luciferase are shown by SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

The nucleotide sequence and amino acid sequence of Renilla luciferase mutant described in Inouye & Shimomura, Biochem. Biophys. Res. Commun., 233 (1997) 349-353 are shown by SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

In one embodiment of the present invention, Renilla reniformis-derived luciferase, e.g., a protein comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14 is used as the emission-catalyzing enzyme.

In another embodiment of the present invention, a mutant of Renilla luciferase, e.g., a protein comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 20 is used as the emission-catalyzing enzyme. In some embodiments of the present invention, the maximum emission wavelength can be shifted toward the longer wavelength side relative to Renilla reniformis-derived luciferase by using the mutant of Renilla luciferase as the emission-catalyzing enzyme.

Light emits by contacting these emission-catalyzing enzymes with the ν-coelenterazine compound in some embodiments of the present invention. The emission time is usually 0.01 to an hour. However, the emission time may be prolonged or further shortened by selecting conditions.

(2) Detection or Quantification of Calcium Ions

The photoprotein of the present invention obtained as described above is a non-covalent complex of an apoprotein and a peroxide of the ν-coelenterazine compound formed from the ν-coelenterazine compound and molecular oxygen and is a photoprotein (holoprotein) that emits light by the action of calcium ions. Therefore, the photoprotein of the invention can be used for the detection or quantification of calcium ions.

The detection or quantification of calcium ions can be performed, for example, by adding a sample solution directly to a solution of the photoprotein and measuring the luminescence generated. Alternatively, calcium ions can also be detected or quantified by adding a solution of the photoprotein to a sample solution and measuring the luminescence generated. The photoprotein above may be formed by previously contacting an aqueous apoprotein solution with the ν-coelenterazine compound of the present invention prior to its addition to the assay system for the detection or quantification of calcium ions and the resulting photoprotein may be provided for use. The photoprotein comprising an apoprotein and a peroxide of the ν-coelenterazine compound may also be formed in the assay system by contacting the apoprotein with the ν-coelenterazine compound. The photoprotein formed is a complex (photoprotein) of the apoprotein and the peroxide of the ν-coelenterazine compound of the invention. The above complex (i.e., the photoprotein of the present invention) emits light dependently on the calcium ion concentration.

The detection or quantification of calcium ions can be performed by measuring the luminescence of the photoprotein of the invention through the action of calcium ions, using a luminometer. Luminometers which may be used include commercially available instruments, such as a Centro LB 960 (manufactured by Berthold, Inc.), etc. The calcium ion concentration can be quantitatively determined by preparing a luminescence standard curve for known calcium ion concentrations using the photoprotein.

The ν-coelenterazine compound of the present invention may also be used for the detection of changes in the intracellular calcium ion concentration under the physiological conditions by preparing the photoprotein comprising an apoprotein and a peroxide of the ν-coelenterazine compound and injecting the photoprotein directly into cells by means of microinjection, etc.

The ν-coelenterazine compound of the present invention may also be used to produce the photoprotein, which is performed, in addition to the injection using techniques such as microinjection, by intracellularly expressing a gene for the apoprotein (a polynucleotide encoding the apoprotein) to produce the protein in the cells and adding the ν-coelenterazine compound of the present invention to the resulting apoprotein from the external cells.

Using the photoprotein of the present invention thus introduced into cells or produced in cells, changes in the intracellular calcium ion concentration caused by external stimulation (e.g., stimulation with receptor-associated drugs, etc.) can also be determined.

(3) Use as Reporter Protein, etc. Utilizing Luminescence

The photoprotein of the present invention can also be used as a reporter protein to determine the transcription activity of a promoter, etc. A polynucleotide encoding an apoprotein is fused to a target promoter or other expression control sequence (e.g., an enhancer, etc.) to construct a vector. The vector described above is transfected to a host cell, and the ν-coelenterazine compound of the present invention is brought in contact with the cell. By detecting the luminescence from the photoprotein of the present invention, the activity of the target promoter or other expression control sequence can be determined. As used herein, the term “contact” means that a host cell and the ν-coelenterazine compound are allowed to be present in the same culture system/reaction system, and includes, for example, addition of the ν-coelenterazine compound to a culture container charged with a host cell, mixing of a host cell with the ν-coelenterazine compound, culture of a host cell in the presence of the ν-coelenterazine compound, and the like.

The ν-coelenterazine compound of the present invention can be used to determine the transcription activity of a promoter, etc. For example, a polynucleotide encoding the emission-catalyzing enzyme is fused to a target promoter or other expression control sequence (e.g., an enhancer, etc.) to construct a vector. The vector described above is transfected to a host cell, and the ν-coelenterazine compound of the present invention is contacted with the cell. By detecting the luminescence from the ν-coelenterazine compound of the present invention, the activity of the target promoter or other expression control sequence can be determined. As used herein, the term “contact” means that a host cell and the ν-coelenterazine compound of the present invention are allowed to be present in the same culture system/reaction system, and includes, for example, addition of the ν-coelenterazine compound to a culture container charged with a host cell, mixing of a host cell with the ν-coelenterazine compound, culture of a host cell in the presence of the ν-coelenterazine compound, and the like. The emission-catalyzing enzyme is described above and is at least one luciferase selected from the group consisting of Renilla luciferase, Oplophorus luciferase, Gaussia luciferase and mutants thereof, and preferably, Renilla luciferase mutants.

The present invention further provides a kit used for the measurement of transcription activity of a promoter, etc. In some embodiments of the present invention, the kit comprises the ν-coelenterazine compound of the present invention and the emission-catalyzing enzyme. In another embodiment of the present invention, the kit comprises the photoprotein of the present invention and the coelenterazine compound. Reagent such as the coelenterazine compound, the emission-catalyzing enzyme, etc. may be dissolved in a suitable solvent and prepared to be suitable for storage. The solvent which may be used is at least one selected from the group consisting of water, ethanol, various buffer solutions, and the like. The kit may additionally comprise, if necessary, at least one selected from the group consisting of a container designed therefor, other necessary accessories and an instruction manual, and the like.

(4) Use as Detection Marker, etc. Utilizing Luminescence

The photoprotein of the present invention can be used as a marker for detection by luminescence. The detection marker of the present invention can be used to detect a target substance in, e.g., immunoassay, hybridization assay, etc. The photoprotein of the present invention can be used in the form bound to a target substance (protein, nucleic acid, etc.) in a conventional manner including chemical modification. Detection methods using such a detection marker can be performed in a conventional manner. The detection marker of the present invention can also be used to determine the distribution of a target substance by expressing the marker, e.g., as a fusion protein of the apoprotein and the target substance, then inserting the fusion protein into cells by means of microinjection or the like and contacting them with the ν-coelenterazine compound of the present invention thereby to produce the photoprotein of the present invention. As used herein, the term “contact” means that cells and the ν-coelenterazine compound of the present invention are allowed to be present in the same culture system/reaction system, and includes, for example, addition of the ν-coelenterazine compound of the present invention to a culture container charged with cells, mixing of cells with the ν-coelenterazine compound of the present invention, culture of host cells in the presence of the ν-coelenterazine compound of the present invention, and the like.

The present invention further provides a method for detection of a target substance in, e.g., immunoassay, hybridization assay, etc., which comprises using the ν-coelenterazine compound and the emission-catalyzing enzyme. In this case, the emission-catalyzing enzyme can be used in the form bound to the target substance (protein, nucleic acid, etc.) in a conventional manner, including chemical modification. Such a detection method using the detection marker can be performed in a conventional manner. The detection marker can also be used to determine the distribution of the target substance by expressing the marker, e.g., as a fusion protein of the emission-catalyzing enzyme and the target substance, then inserting the fusion protein into cells by means of microinjection or the like and contacting it with the ν-coelenterazine compound of the present invention. As used herein, the term “contact” means that a cell and the ν-coelenterazine compound of the present invention are allowed to be present in the same culture system/reaction system, and includes, for example, addition of the ν-coelenterazine compound of the present invention to a culture container charged with a cell, mixing of a cell with the ν-coelenterazine compound of the present invention, culture of a host cell in the presence of the ν-coelenterazine compound of the present invention, and the like. The emission-catalyzing enzyme is the same as described above and is, for example, at least one luciferase selected from the group consisting of Renilla luciferase, Oplophorus luciferase and Gaussia luciferase.

The distribution of these target substances, etc. can be determined by a method for detection such as luminescence imaging, etc. The apoprotein may also be used after its expression in cells, in addition to the insertion into cells by means of microinjection, etc.

The present invention further provides a kit used for the detection of a target substance in an immunoassay, hybridization assay, etc. The kit in some embodiments of the present invention comprises the photoprotein of the present invention and the coelenterazine compound. The kit in another embodiment of the present invention comprises the ν-coelenterazine compound of the present invention and the emission-catalyzing enzyme. Reagent such as the coelenterazine compound, the emission-catalyzing enzyme, etc. may be dissolved in a suitable solvent and prepared to be suitable for storage. The solvent which may be used is at least one selected from the group consisting of water, ethanol, various buffer solutions, and the like. The kit may additionally comprise, if necessary, at least one selected from the group consisting of a container designed therefor, other necessary accessories and an instruction manual, and the like.

(5) Material for Amusement Supplies

The complex (photoprotein of the present invention) comprising the apoprotein and a peroxide of the ν-coelenterazine compound of the present invention emits light only by binding to a trace of calcium ions. The photoprotein of the present invention can thus be preferably used as a luminescence material for amusement supplies. Examples of such amusement supplies are luminescent soap bubbles, luminescent ice, luminescent candies, luminescent color paints, etc. The amusement supplies of the present invention can be prepared in a conventional manner.

(6) Bioluminescence Resonance Energy Transfer (BRET) Method

In some embodiments of the present invention, the ν-coelenterazine compound emits light by the action of the emission-catalyzing enzyme as described above, and can be used for analysis methods including an analysis of physiological functions, an analysis of (or assay for) enzyme activities, etc., based on the principle of intermolecular interaction by the bioluminescence resonance energy transfer (BRET) method. The photoprotein of the present invention can also be used for analyses including an analysis of biological functions, an assay for enzyme activities, etc., based on the principle of intermolecular interaction by the bioluminescence resonance energy transfer (BRET) method.

For example, using the ν-coelenterazine compound and the emission-catalyzing enzyme in some embodiments of the present invention as donor proteins and as an acceptor protein an organic compound or a fluorescent protein, the interaction between the proteins can be detected by generating bioluminescence resonance energy transfer (BRET) between them. As used herein, the emission-catalyzing enzyme is the same as given before and is, for example, at least one luciferase selected from the group consisting of Renilla luciferase, Oplophorus luciferase and Gaussia luciferase.

Alternatively, using the photoprotein of the invention as a donor protein and an organic compound or a fluorescent protein as an acceptor protein, the interaction between the proteins can be detected by generating bioluminescence resonance energy transfer (BRET) between them. In a certain embodiment of the present invention, the organic compound used as an acceptor protein is Hoechist 3342, Indo-1, DAP1, etc. In another embodiment of the present invention, the fluorescent protein used as an acceptor protein is a green fluorescent protein (GFP), a blue fluorescent protein (BFP), a mutant GFP fluorescent protein, phycobilin, etc. In a preferred embodiment of the present invention, the physiological functions to be analyzed include an orphan receptor (in particular, G-protein conjugated receptor), apoptosis, transcription regulation, etc. by gene expression. Still in a preferred embodiment of the present invention, the enzyme to be analyzed is protease, esterase, kinase, etc.

Analysis of the physiological functions by the BRET method may be performed by known methods, for example, by modifications of the methods described in Biochem. J. 2005, 385, 625-637, Expert Opin. Ther. Targets, 2007 11: 541-556, etc. Assay for the enzyme activities may also be performed by known methods, for example, by modifications of the methods described in Nat. Methods 2006, 3:165-174, Biotechnol. J. 2008, 3: 311-324, etc.

The present invention further provides a kit used for the analysis methods described above. The kit comprises the ν-coelenterazine compound of the present invention, the emission-catalyzing enzyme and the organic compound and/or the fluorescent protein. Reagents such as the ν-coelenterazine compounds, the emission-catalyzing enzymes, organic compounds, fluorescent proteins, etc. may be dissolved in a suitable solvent and prepared to be suitable for storage. The solvent which may be used is at least one selected from the group consisting of water, ethanol, various buffer solutions, and the like. The kit may additionally comprise, if necessary, at least one selected from the group consisting of a container designed therefor, other necessary accessories and an instruction manual, and the like.

All literatures and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, irrespective of their purposes. The disclosure of the specification, the claims, abstract and drawings of Japanese Application JP2011-050487 filed on Mar. 8, 2011, based upon which the present application claims the benefit of priority, are entirely incorporated herein by reference.

The objects, characteristics and advantages of the present invention as well as the idea thereof are apparent to those skilled in the art from the descriptions given herein, and those skilled in the art can easily implement the present invention. It is to be understood that the best mode to carry out the invention and specific examples are to be taken as preferred embodiments of the present invention. These descriptions are only for illustrative and explanatory purposes and are not intended to restrict the invention thereto. It is apparent to those skilled in the art that various modifications may be made based on the descriptions given herein within the intent and scope of the present invention disclosed herein.

EXAMPLES

Hereinafter, the present invention will be illustrated more specifically with reference to SYNTHESIS EXAMPLES and EXAMPLES, but it should be understood that the invention is not deemed to be limited to these SYNTHESIS EXAMPLES and EXAMPLES.

SYNTHESIS EXAMPLES Overview

The process for producing the ν-coelenterazine compound illustratively shown in these SYNTHESIS EXAMPLES is a highly flexible and highly efficient process which is applicable to the production of various ν-coelenterazine analogues.

More specifically, in these SYNTHESIS EXAMPLES, commercially available 2-chloro-6-aminopyrazine 3 was first dibrominated to synthesize known Compound 4 (WO 2003/059893). The compound was subjected to the Negishi coupling with benzyl zinc reagent to introduce benzyl into Compound 4, whereby Compound 5 could be obtained. Next, the Suzuki-Miyaura coupling was performed between Compound 5 and boronic acid 6 so that Compound 5 was arylated at the 5-position selectively to give Compound 7. Then, the 6-position of Compound 7 was vinylated through the Stille coupling with vinyl tin to give Compound 8 successfully. It is considered that the respective substituents could be introduced with high position selectivity by the effect of unprotected amino group in these 3 types of the coupling reactions.

The formyl group of Compound 8 obtained was further methylated to give Compound 9. After the amino group in Compound 9 was protected with acetyl, the ring-closing metathesis reaction was performed using a Hoveyda-Grubbs second generation catalyst, whereby tricyclic aromatic Compound 11 could be efficiently obtained. In this case, though the yield was low, the ring-closing metathesis reaction could proceed to give the corresponding cyclic product 12 from Compound 9 with the amino being unprotected. However, it was found that the ring closing reaction proceeded more efficiently with an acyl-protected compound.

Subsequently, the methyl group in Compound 11 was removed by boron tribromide. The resulting Compound 13 was heated in methanol in the presence of a small quantity of sodium hydrogencarbonate for deprotection of acetyl to give ν-coelenteramine 4. For removal of the methyl group in Compound 12 wherein the amino group is not protected with acetyl group, boron tribromide could not be used but by heating with pyridine hydrochloride, ν-coelenteramine 14 could be obtained while the yield was low.

Finally, ν-coelenteramine 14 was condensed and cyclized with the ketacetal 15. Thus, ν-CTZ (2) could be synthesized.

This synthetic route is as short as 8 to 10 steps from the commercially available compound. In addition, the stability is expected to be improved by varying the substituent on the benzene ring of the ketacetal used in the final step, and a variety of ν-CTZ analogues can be synthesized in a simple manner (cf., e.g., the equation below).

In the future it is expected that ν-CTZ compounds suitable for diverse applications including near-infra red luminescent imaging can be newly produced by using this process for synthesis.

Materials and Methods (1) Chromatography

Thin layer chromatography (TLC) for analysis was performed on a glass plate (MERCK 5715, silica gel 60 F₂₅₄) previously coated with silica gel. The spots were detected under a UV lamp (254 nm or 365 nm) by adsorbing iodine, dipping in an aqueous anisaldehyde solution and charring on a hot plate.

For preparative flush column chromatography, silica gels (Kanto Chemical Co., Inc., 37563-85, silica gel 60 N (spherical, neutral), particle size 45-50 μm or Kanto Chemical Co., Inc., 37565-85, silica gel 60 N (spherical, neutral), particle size 63-210 μm) were used. For purification of the CTZ analogues, however, silica gels (Kanto Chemical Co., Inc., 37562-79, silica gel 60 N (spherical), particle size 40-50 μm or Kanto Chemical Co., Inc., 37558-79, silica gel 60 N (spherical), particle size 100-210 μm) were used.

(2) Nuclear Magnetic Resonance (NMR) Spectrum

¹H Nuclear magnetic resonance (NMR) spectrum (400 MHz) was determined on an AVANCE 400 or AVANCE 500 nuclear magnetic resonance apparatus manufactured by Bruker, Inc. Chemical shifts (δ) were expressed as values relative to the peaks from tetramethylsilane ((CH₃)₄Si) (measured in CDCl₃; 0 ppm) or the peaks from non-deuterated solvent for analysis (measured in CD₃OD; 3.31 ppm; measured in DMSO-d₆; 2.49 ppm) as an internal standard. Abbreviations s, d and m used for the signal splitting patterns represent singlet, doublet and multiplet, respectively.

¹³C Nuclear magnetic resonance spectrum (100 MHz) was determined by a AVANCE 400 nuclear magnetic resonance apparatus manufactured by Bruker, Inc. Chemical shifts (δ) were expressed as values relative to the peaks from carbon in the solvent for analysis (measured in CDCl₃; 77.0 ppm, measured in CD₃OD; 49.0 ppm) as an internal standard.

(3) Infrared Absorption (IR) Spectrum

IR spectra were determined by the diffuse reflection method using a SHIMAZU IR Prestige-21 spectrophotometer equipped with DRS-8000, manufactured by Shimadzu Corporation.

(4) Mass Spectrometry

High resolution mass spectrometry (HRMS) was performed by the electrospray ionization method (ESI) with a Bruker micrOTOF manufactured by Bruker, Inc.

(5) Chemical Reagent

The reagents were used without further treatment unless otherwise indicated. The solvents for the reactions, extractions and chromatography were acetonitrile, ethyl acetate, n-hexane, anhydrous tetrahydrofuran (THF), toluene, anhydrous pyridine, 1,2-dichloroethane, anhydrous dichloromethane, methanol and ethanol, all commercially available, and used without further treatment. Unless otherwise indicated, mixing ratios in the solvent mixtures used are based on “v/v.”

The reaction reagents below were used. 2-Amino-6-chloropyrazine(Cat. No. 1650) purchased from Matrix Scientific, N-bromosuccinimide (Cat. No. 025-07235), 2-formyl-4-methoxyphenylboronic acid (Cat. No. 328-99903), potassium fluoride (Cat. No. 169-03765), tetra-n-butylammonium chloride (Cat. No. T0054), methyltriphenylphosphonium bromide (Cat. No. 138-11961), 4-(dimethylamino)pyridine (Cat. No. 042-19212), acetyl chloride (Cat. No. 011-0053), conc. hydrochloric acid (Cat. No. 080-01066) and pyridinium hydrochloride (Cat. No. 163-11931) purchased from Wako Pure Chemical Co., Ltd., zinc chloride (1.0 M in diethyl ether)(Cat. No. 276839), benzyl magnesium chloride (2.0 M in THF)(Cat. No. 225916), dichlorobis(triphenylphosphine) palladium(II) (Cat. No. 412740), 2-(di-tent-butylphosphino)-1-phenylindole (Cat. No. 672343), tributyl(vinyl)tin (Cat. No. 271438), Hoveyda-Grubbs second generation catalyst (Cat. No. 569755) and boron tribromide (1.0 M in dichloromethane)(Cat. No. 211222) purchased from Aldrich, Inc., allyl palladium(II) chloride dimer (Cat. No. A1479) purchased from Tokyo Chemical Industry Co., Ltd., n-butyl lithium (1.65 M in n-hexane (Cat. No. 04937-25) purchased from Kanto Chemical Co., Inc.; were used in the reactions without further treatment.

SYNTHESIS EXAMPLE 1: 2-Amino-3,5-dibromo-6-chloropyrazine (4)

To a solution of 2-amino-6-chloropyrazine (3) (8.00 g, 61.8 mmol) in acetonitrile (80 mL) was gradually added N-bromosuccinimide (NBS) (27.5 g, 155 mmol) at 0° C. After elevating to room temperature, the mixture was stirred overnight (18 hours). To the mixture was added water and the product was extracted with diethyl ether (×3). The combined organic extract was washed successively with water (×1) and brine (×1), followed by drying over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=3/1) to give 2-amino-3,5-dibromo-6-chloropyrazine (4) (16.8 g, 58.5 mmol, 94.7%) as a yellow solid. TLC R_(f)=0.31 (n-hexane/ethyl acetate=4/1); ¹H NMR (500 MHz, CDCl₃) δ 5.14 (s, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 120.7, 122.0, 146.1, 151.0.

SYNTHESIS EXAMPLE 2: 2-Amino-3-benzyl-5-bromo-6-chloropyrazine (5)

Under an argon atmosphere, to anhydrous THF (40 mL) was added zinc chloride (1.0 M in diethyl ether) (23.7 mL, 23.7 mmol), and to the mixture was added benzyl magnesium chloride (2.0 M in THF) (10.4 mL, 20.8 mmol). After stirring at room temperature for 30 minutes, to the mixture were successively added dichlorobis(triphenylphosphine)palladium(II) (488 mg, 695 μmol) and 2-amino-3,5-dibromo-6-chloropyrazine (4) (4.00 g, 13.9 mmol) at room temperature. The mixture was stirred overnight (15 hours and a half) at room temperature as it was. After to the mixture was added water, the product was extracted with ethyl acetate (×3). The combined organic extract was washed successively with water (×1) and brine ('1), followed by drying over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=5/1) to give 2-amino-3-benzyl-5-bromo-6-chloropyrazine (5) (3.64 g, 12.2 mmol, 87.6%) as a yellow solid. TLC R_(f)=0.25 (n-hexane/ethyl acetate=4/1); ¹H NMR (500 MHz, CDCl₃) δ 4.08 (s, 2H), 4.50 (s, 2H), 7.19-7.24 (m, 2H), 7.27-7.37 (m, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 40.1, 123.8, 127.5, 128.4, 129.3, 135.3, 139.5, 144.9, 151.3.

SYNTHESIS EXAMPLE 3: 2-(5-Amino-6-benzyl-3-chloropyrazin-2-yl)-5-methoxybenzaldehyde (7)

Under an argon atmosphere, to a solution of allylpalladium(II) chloride dimer (160 mg, 437 μmol) in anhydrous THF (30 mL) was added 2-(di-tert-butylphosphino)-1-phenylindole (295 mg, 874 μmol) at room temperature, followed by stirring at room temperature for 10 minutes. Subsequently, to the mixture were successively added 2-amino-3-benzyl-5-bromo-6-chloropyrazine (5) (2.60 g, 8.74 mmol), 2-formyl-4-methoxyphenylboronic acid (6) (3.14 g, 17.4 mmol), potassium fluoride (2.60 g, 44.8 mmol) and water (160 μL, 8.88 mmol) at room temperature. The mixture was stirred overnight (14 hours) at room temperature without further treatment. After to this was added water, the product was extracted with ethyl acetate (×3). The combined organic extract was washed successively with water (×1) and brine (×1), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=3/1) to give 2-(5-amino-6-benzyl-3-chloropyrazin-2-yl)-5-methoxybenzaldehyde (7) (2.37 g, 6.70 mmol, 76.9%) as a reddish brown solid. TLC R_(f)=0.27 (n-hexane/ethyl acetate=3/1); ¹H NMR (500 MHz, CDCl₃) δ 3.92 (s, 3H), 4.13 (s, 2H), 4.66 (s, 2H), 7.21-7.25 (m, 3H), 7.27-7.31 (m, 1H), 7.32-7.37 (m, 2H), 7.51-7.55 (m, 2H), 9.92 (s, 1H).; ¹³C NMR (126 MHz, CDCl₃) δ 40.4, 55.7, 111.2, 120.7, 127.4, 128.4(2C), 129.2(2C), 132.5, 132.7, 135.7, 135.8, 137.8, 139.1, 144.1, 151.5, 160.0, 191.0.

SYNTHESIS EXAMPLE 4: 2-(5-Amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde (8)

Under an argon atmosphere, to a solution of 2-(5-amino-6-benzyl-3-chloropyrazin-2-yl)-5-methoxybenzaldehyde (7) (2.28 g, 6.44 mmol) in deaerated toluene (30 mL) were successively added dichlorobis(triphenylphosphine)palladium (II) (226 mg, 322 μmol), tributyl(vinyl)tin (3.75 mL, 12.8 mmol) and tetra-n-butylammonium chloride (3.50 g, 12.6 mmol) at room temperature. The mixture was heated to reflux for 1.5 hours (oil bath temperature at 110° C.). After cooling to room temperature, to the mixture was added water and the product was extracted with ethyl acetate (×3). The combined organic extract was washed successively with water (×1) and brine (×1) followed by drying over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (silica gel mixed with 10 wt % potassium fluoride, n-hexane/ethyl acetate=3/1) to give 2-(5-amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde (8) (1.24 g, 3.59 mmol, 55.5%) as a reddish brown oily substance. TLC R_(f)=0.27 (n-hexane/ethyl acetate=3/1); ¹H NMR (500 MHz, CDCl₃) δ 3.92 (s, 3H), 4.15 (s, 2H), 4.50 (s, 2H), 5.39 (dd, 1H, J=2.0, 11.0 Hz), 6.33 (dd, 1H, J=2.0, 17.0 Hz), 6.57 (dd, 1H, J=11.0, 17.0 Hz), 7.20-7.28 (m, 4H), 7.30-7.36 (m, 2H), 7.41 (d, 1H, J=8.5 Hz), 7.55 (d, 1H, J=3.0 Hz), 9.88 (s, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 41.0, 55.7, 110.6, 120.5, 121.0, 127.2, 128.5 (2C), 129.1 (2C), 132.3, 132.9, 134.4, 136.1, 136.5, 138.7, 140.2, 145.2, 151.5, 159.8, 191.5.

SYNTHESIS EXAMPLE 5: 2-Amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine (9)

Under an argon atmosphere, to a suspension of methyltriphenylphosphonium bromide (1.29 g, 3.61 mmol) in anhydrous THF (5 mL) was gradually dropwise added n-butyl lithium (1.65 M in n-hexane) (1.70 mL, 2.81 mmol) at 0° C. The mixture was stirred at 0° C. for 15 minutes as it was. To the mixture was added 2-(5-amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde (8) (871 mg, 2.52 mmol) at 0° C. After elevating to room temperature, stirring was continued for 1.5 hours. After to this was added water, the product was extracted with ethyl acetate (×3). The combined organic extract was washed successively with water (×1) and brine (×1), followed by drying over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=3/1) to give 2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine (9) (570 mg, 1.66 mmol, 65.8%) as a white solid. TLC R_(f)=0.37 (n-hexane/ethyl acetate=3/1); ¹H NMR (500 MHz, CDCl₃) δ 3.88 (s, 3H), 4.16 (s, 2H), 4.39 (s, 2H), 5.18 (dd, 1H, J=1.0, 10.5 Hz), 5.30 (dd, 1H, J=2.0, 10.5 Hz), 5.67 (dd, 1H, J=1.0, 17.5 Hz), 6.25 (dd, 1H, J=2.0, 17.5 Hz), 6.47 (dd, 1H, J=10.5, 17.5 Hz), 6.50 (dd, 1H, J=10.5, 17.5 Hz), 6.90 (dd, 1H, J=2.5, 8.0 Hz), 7.20-7.26 (m, 5H), 7.29-7.35 (m, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 41.2, 55.6, 110.5, 113.8, 115.4, 119.3, 127.2, 128.7 (2C), 129.2 (2C), 129.8, 132.1, 133.0, 135.1, 137.0, 138.3, 140.3, 141.9, 144.8, 151.4, 159.8.

SYNTHESIS EXAMPLE 6: N-(3-Benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazin-2-yl)acetamide (10)

Under an argon atmosphere, to a solution of 2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine (9) (100 mg, 292 μmol) in anhydrous pyridine (20 mL) were successively added 4-(dimethylamino)pyridine (DMAP)(3.0 mg, 25 μmol) and acetyl chloride (65.0 μL, 911 μmol) at room temperature. The mixture was heated at 60° C. and stirred for an hour. After cooling to room temperature, to the mixture was added water and the product was extracted with ethyl acetate (×3). The combined organic extract was washed successively with water (×1) and brine (×1), followed by drying over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=3/1) to give

N-(3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazin-2-yl)acetamide (10) (89.5 mg, 232 μmol, 79.7%) as a yellow solid. TLC R_(f)=0.17 (n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz, CDCl₃) δ 2.33 (s, 3H), 3.89 (s, 3H), 4.28 (s, 2H), 5.19 (dd, 1H, J=1.0, 10.8 Hz), 5.39 (dd, 1H, J=2.0, 10.8 Hz), 5.66 (dd, 1H, J=1.0, 17.2 Hz), 6.30 (dd, 1H, J=2.0, 17.2 Hz), 6.45 (dd, 1H, J=10.8, 17.2 Hz), 6.52 (dd, 1H, J=10.8, 17.2 Hz), 6.94 (dd, 1H, J=2.6, 8.4 Hz), 7.19-7.24 (m, 5H), 7.27-7.32 (m, 2H).

SYNTHESIS EXAMPLE 7: N-(2-Benzyl-8-methoxybenzo[f]quinoxalin-3-yl)acetamide (11)

Under an argon atmosphere, to a solution of Hoveyda-Grubbs second generation catalyst (61.3 mg, 97.8 μmol) in 1,2-dichloromethane (150 mL) was added N-(3 -benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazin-2-yl)acetamide (10) (348 mg, 903 μmol) at room temperature. The mixture was then heated to 90° C. and stirred overnight (17 hours). After cooling to room temperature and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=4/3→1/1) to give N-(2-benzyl-8-methoxybenzo[f]quinoxalin-3-yl)acetamide (11) (277 mg, 775 μmol, 85.8%) as a white solid. TLC R_(f)=0.27 (n-hexane/ethyl acetate=1/1); ¹H NMR (400 MHz, CDCl₃) δ 2.33 (s, 3H), 4.00 (s, 3H), 4.50 (s, 2H), 7.24-7.34 (m, 5H), 7.38 (dd, 1H, J=2.6, 8.8 Hz), 7.42 (br, 1H), 7.90 (d, 1H, J=8.8 Hz), 7.93 (d, 1H, J=8.8 Hz), 9.01 (d, 1H, J=8.8 Hz).

SYNTHESIS EXAMPLE 8: N-(2-Benzyl-8-hydroxybenzo[f]quinoxalin-3-yl)acetamide (13)

Under an argon atmosphere, to a suspension of N-(2-benzyl-8-methoxybenzo[f]quinoxalin-3-yl)acetamide (60.0 mg, 168 μmol) in anhydrous dichloromethane (2 mL) was added boron trifluoride (1.0 M in dichloromethane) (840 μL, 840 μmol) at 0° C. After elevating to room temperature, the mixture was stirred for 3 hours. To the mixture was then added a solution of saturated sodium hydrogencarbonate, and the product was extracted with ethyl acetate (×3). The combined organic extract was washed successively with water (×1) and brine (×1), followed by drying over anhydrous sodium sulfate. Filtration and concentration under reduced pressure gave N-(2-benzyl-8-hydroxybenzo[f]quinoxalin-3-yl)acetamide (13) as a crude yellow solid product. The product was used for the subsequent reaction without any further purification. TLC R_(f)=0.36 (n-hexane/ethyl acetate=1/2); ¹H NMR (500 MHz, CD₃OD) δ 2.17 (s, 3H), 4.50 (s, 2H), 7.20-7.32 (m, 9H), 7.78 (d, 1H, J=9.0 Hz), 7.96 (d, 1H, J=9.0 Hz), 9.44 (d, 1H, J=7.5 Hz).

SYNTHESIS EXAMPLE 9: 3-Amino-2-benzylbenzo[f]quinoxalin-8-ol (14, ν-coelenteramine)

A solution of the obtained crude product of N-(2-benzyl-8-hydroxybenzo[f]quinoxalin-3-yl)acetamide (13) in methanol (20 mL) was heated to 65° C. and stirred overnight (14 hours). After cooling to room temperature, the solution was concentrated under reduced pressure and the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=1/1→1/10) to give 3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14, ν-coelenteramine) (51.3 mg, 170 mmol, quant., two steps) as an ocherous solid. TLC R_(f)=0.34 (n-hexane/ethyl acetate=1/1); ¹H NMR (500 MHz, CD₃OD) δ 4.33 (s, 2H), 7.16-7.20 (m, 2H), 7.21-7.26 (m, 1H), 7.30-7.38 (m, 5H), 7.50 (d, 1H, J=9.0 Hz), 7.76 (d, 1H, J=9.0 Hz), 8.77 (d, 1H, J=9.0 Hz).

SYNTHESIS EXAMPLE 10: 3-Amino-2-benzyl-8-methoxyaline (12)

Under an argon atmosphere, to a solution of Hoveyda-Grubbs second generation catalyst (84.5 mg, 135 μmol) in 1,2-dichloroethane (100 mL) was added 2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine (9) (585 mg, 1.70 mmol) at room temperature. The mixture was then heated to 90° C. and stirred overnight (17 hours). After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=3/1→2/1) to give 3-amino-2-benzyl-8-methoxybenzo[f]quinoxaline (12) as a white solid (136 mg, 431 μmol, 25.3%). TLC R_(f)=0.64 (n-hexane/ethyl acetate=1/1); NMR (500 MHz, CDCl₃) δ 3.98 (s, 3H), 4.39 (s, 2H), 4.63 (s, 2H), 7.21-7.38 (m, 7H), 7.63 (d, 1H, J=9.0 Hz), 7.85 (d, 1H, J=9.0 Hz), 9.00 (d, 1H, J=9.0 Hz); ¹³C NMR (126 MHz, CDCl₃) δ 41.8, 55.4, 107.5, 118.1, 124.9, 125.5, 125.8, 127.2, 128.7 (2C), 129.1 (2C), 130.1, 132.7, 134.6, 136.6, 138.9, 143.1, 151.2, 158.5.

SYNTHESIS EXAMPLE 11: 3-Amino-2-benzylbenzo[f]quinoxalin-8-ol (14,ν-coelenteramine)(Synthesis from 12)

A mixture of 3-amino-2-benzyl-8-methoxybenzo[f]quinoxaline (12) (125 mg, 396 μmol) and pyridinium hydrochloride (2.50 g, 21.6 mmol) was stirred at 190° C. (oil bath temperature) for 30 minutes and at 210° C. (oil bath temperature) for further 30 minutes. After cooling to room temperature, to the mixture was added water, the product was extracted with ethyl acetate (×3). The combined organic extract was washed successively with water (×1) and brine (×1), followed by drying over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=1/1→1/10) to give 3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14, ν-coelenteramine) (55.5 mg, 184 μmol, 46.5%) as an ocherous solid.

SYNTHESIS EXAMPLE 12: ν-Coelenterazine (2, ν-CTZ)

Under an argon atmosphere, to a solution of 3-(4-(tert-butyldimethylsilyloxy)phenyl)-1,1-diethoxypropan-2-one (15) (101 mg, 286 μmol) in ethanol (2 mL) and water (0.4 mL) was added 3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14) (55.5 mg, 184 μmol). After cooling to 0° C., to the mixture was further added conc. hydrochloric acid (0.20 mL). The mixture was heated to 80° C. and stirred overnight (18 hours). After cooling to room temperature and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=1/1→1/2→ethyl acetate alone) in an argon flow to give ν-coelenterazine (2, ν-CTZ) (10.9 mg, 24.4 μmol, 13.2%) as ocherous powders. TLC R_(f)=0.50 (n-hexane/ethyl acetate=1/1); ¹H NMR (500 MHz, CD₃OD) δ 4.08 (s, 2H), 4.51 (s, 2H), 6.69-6.72 (AA′BB′, 2H), 7.17-7.24 (m, 5H), 7.29 (t, 2H, J=7.5 Hz), 7.43 (d, 2H, J=7.5 Hz), 7.63 (d, 1H, J=8.5 Hz), 8.40 (br, 1H), 9.26 (br, 1H).

SYNTHESIS EXAMPLE 13: cf3-ν-coelenterazine (17, cf3-ν-CTZ)

Under an argon atmosphere, to a solution of 3-(4-(trifluoromethyl)phenyl)propan-1,1-diethoxy-2-one (16) (86.9 mg, 299 μmol) in ethanol (2 mL) and water (0.2 mL) was added 3-amino-2-benzylbenzo[f]quinoxalin-8-ol (14) (60.2 mg, 200 μmol). After cooling to 0° C., to the mixture was further added conc. hydrochloric acid (0.10 mL). The mixture was heated to 80° C. and stirred overnight (13 hours). After cooling to room temperature and concentration under reduced pressure, the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate=1/1→1/2→ethyl acetate alone) in an argon flow to give cf3-ν-coelenterazine (17, cf3-ν-CTZ) (28.6 mg, 57.3 μmol, 28.7%) as yellow powders. TLC R_(f)=0.24 (n-hexane/ethyl acetate=1/2); ¹H NMR (500 MHz, CD₃OD) δ 4.26 (s, 2H), 4.51 (s, 2H), 7.18-7.25 (m, 3H), 7.28-7.33 (m, 2H), 7.40-7.44 (AA′BB′, 2H), 7.54-7.66 (m, 5H), 8.29 (br, 1H), 9.17 (br, 1H).

EXAMPLE 1: Construction of Expression Vector 1) Construction of Renilla Luciferase Expression Vector pCold-RL

Novel expression vector pCold-RL was constructed by the following procedures. Using the vector pHis-RL having Renilla luciferase gene, described in Inouye & Shimomura, Biochem. Biophys. Res. Commun., 233 (1997) 349-353, as a template, PCR was performed (cycle conditions: 25 cycles; 1 min/94° C., 1 min/50° C., 1 min/72° C.) with a PCR kit (manufactured by Takara Bio Inc.) using two PCR primers, RL-17N/SacI (5′ gcc GAG CTC ACT TCG AAA GTT TAT GAT CC 3′: SEQ ID NO: 21) and RL-18C/XhoI (5′ cgg CTC GAG TTA TTG TTC ATT TTT GAG AA 3′: SEQ ID NO: 22). After purification by a PCR purification kit (manufactured by Qiagen Inc.) and digestion with the restriction enzymes Sad and XhoI, the resulting fragment was inserted into the SacI/XhoI restriction enzyme site of vector pCold-II (manufactured by Takara Bio Inc., GenBank: AB186389) to give Renilla luciferase expression vector pCold-RL. The nucleotide sequence of the insert DNA was confirmed by a DNA sequencer (manufactured by ABI Inc.).

2) Construction of Mutant Renilla Luciferase-547 Expression Vector pCold-hRL-547 to Shift the Emission to Longer Wavelength

The novel expression vector pCold-hRL-547 having mutant Renilla luciferase-547 gene (hRL-547) was constructed by the following procedures. The vector pCR2.1-hRL-547 plasmid having mutant Renilla luciferase-547 gene was constructed by the chemical synthesis and PCR procedures. The mutant Renilla luciferase-547 gene was constructed as follows: the construction of pCR2.1-hRL-547 involved designing based on RLuc8.6-547 described in Nature Methods, 4 (2007) 641-643, synthesizing by a combination of conventional chemical synthesis with PCR (Operon Inc.) and cloning into the Asp718/EcoRI restriction enzyme site of pCR2.1 (Invitrogen Inc.). In order to construct the pCold-hRL-547 vector, pCR2.1-hRL-547 was digested with restriction enzymes Asp718/EcoRI and inserted into the Asp718/EcoRI restriction enzyme site of vector pCold-II to construct Renilla luciferase-547 expression vector pCold-hRL-547. The nucleotide sequence of the insert DNA was confirmed with a DNA sequencer (manufactured by ABI Inc.).

The nucleotide sequence of DNA encoding hRL-547 is shown by SEQ ID NO: 19. The amino acid sequence of hRL-547 is shown by SEQ ID NO: 20.

EXAMPLE 2: Purification of Recombinant Protein 1) Expression of Recombinant Protein in Escherichia coli

To express the recombinant protein in Escherichia coli, Renilla luciferase gene expression vector pCold-RL and Renilla luciferase-547 gene expression vector pCold-hRL-547 were used. The vectors were transformed into Escherichia coli BL21 and the resulting transformants were cultured in 10 mL of LB liquid medium (10 g of bactotryptone, 5 g of yeast extract and 5 g of sodium chloride, per liter of water, pH 7.2) supplemented with ampicillin (50 μg/m) and cultured at 37° C. for 16 hours. The cultured LB broth was added to a fresh LB liquid medium of 400 mL×5 (2L in total) followed by incubation at 37° C. for 4.5 hours. After cooling on ice for an hour, 0.2 mM IPTG was added and incubation was continued at 15° C. for 17 hours. After completion of the incubation, the cells were harvested by centrifugation (5,000 rpm, 5 minutes) and used as the starting material for protein extraction.

2) Extraction of Recombinant Protein from Cultured Cells and Nickel Chelate Gel Column Chromatography

The culture cells collected were suspended in 200 mL of 50 mM Tris-HCl (pH7.6). Under cooling on ice, the cells were disrupted by ultrasonication (manufactured by Branson, Sonifier Model Cycle 250) 3 times each for 3 minutes. The cell lysate was centrifuged at 10,000 rpm (12,000×g) at 4° C. for 20 minutes. The soluble fractions obtained were applied onto a nickel-chelate column (Amersham Bioscience, column size: diameter 2.5×6.5 cm) to adsorb the recombinant protein. After washing with 500 mL of 50 mM Tris-HCl (pH 7.6), the objective recombinant protein was eluted with 50 mM Tris-HCl (pH 7.6) containing 0.1M imidazole (manufactured by Wako Pure Chemical Industry). Protein concentration was determined using a commercially available kit (manufactured by Bio-Rad) by the method of Bradford and using bovine serum albumin (manufactured by Pierce) as a standard. The yields in the purification process are shown in TABLE 1.

TABLE 1 shows the purification yields of Renilla luciferase and Renilla luciferase-547.

TABLE 1 Total Specific Total Total Activitiy Activity Volume Protein (×10⁸ rlu) (×10⁸ Purification Process (mL) (mg) (%) (%) rlu/mg) 1) Renilla luciferase Soluble fraction 200 600 (100) 1423.0 (100)   2.37 Dialysis after nickel 40 117.6 (19.6)  834.3 (58.6) 7.09 chelate gel elution 2) Renilla luciferase-547 Soluble fraction 200 480 (100)  79.9 (100) 0.17 Dialysis after nickel 58.5 83.7 (17.4)  61.4 (76.9) 0.73 chelate gel elution

EXAMPLE 3: Measurement of Luminescence Activity

After Renilla luciferase (2.94 ng) or Renilla luciferase-547 (14.3 ng) was dispensed into a 96-well microplate (manufactured by Nunc) in an amount of 10 μL/well, 0.1 mL of 30 mM Tris-HCl (pH 7.6)-10 mM EDTA containing 0.05 μg of coelenterazine or ν-coelenterazine was injected into the wells thereby to initiate the luminescence reaction, and the luminescence intensity was measured using a luminescence plate reader Centro LB960 (manufactured by Berthold). The luminescence intensity was measured for 60 seconds 3 times in 0.1 second intervals, and the maximum luminescence intensity (I_(max)) and the mean (rlu) from luminescence integrated for 60 seconds were expressed in terms of relative activity (%) (TABLE 2).

TABLE 2 shows the luminescence activities of Renilla luciferase and Renilla luciferase-547.

TABLE 2 Maximum Luminescence Intensity I_(max) (Int.) (%) Substrate Renilla Luciferase Renilla Luciferase-547 Coelenterazine 100 (100) 100 (100) v-Coelenterazine 71.8 (47.3)  213 (73.4) cf3-v-Coelenterazine 18.9 (12.3) 16.9 (11.9)

EXAMPLE 4: Measurement of Emission Spectrum

The luminescence reaction was initiated by adding luciferase to 1 mL of 50 mM Tris-HCl (pH 7.6)-10 mM EDTA containing 5 μg (1 μg/82 L) of substrate coelenterazine or ν-coelenterazine dissolved in ethanol. The protein amounts of luciferases used were Renilla luciferase: 1.5 μg and Renilla luciferase-547: 7.2 μg in the case of coelenterazine as substrate. On the other hand, the protein amounts in ν-coelenterazine as substrate were Renilla luciferase: 14.7 μg and Renilla luciferase-547: 35.8 μg. Luminescence spectra were measured in a quartz cell with a 10 mm optical path under the conditions of band width: 20 nm, sensitivity: medium, scan speed: 2000 nm/min and 22-25° C., using a fluorescence spectrophotometer FP-6500 (manufactured by JASCO) with the excitation light source turned off. The emission maximum (λmax, nm) and half bandwidth (nm) were determined from the spectra measured, which are shown in TABLE 3.

TABLE 3 shows the results of analysis of the emission spectra of Renilla luciferase and Renilla luciferase-547.

TABLE 3 Emission Maximum λ_(max) (half bandwidth) (nm) Substrate Renilla luciferase Renilla luciferase-547 Coelenterazine 485.0 (95)  547.0 (124) v-Coelenterazine^(a) 519.0 (105) 593.0 (130) cf3-v-Coelenterazine 525.5 (121) 599.0 (132)

As shown in EXAMPLES above, when ν-coelenterazine was used as a luminescence substrate, the maximum emission wavelength was shifted toward a longer wavelength side relative to coelenterazine. In addition, when cf3-ν-coelenterazine was used as a luminescence substrate, the maximum emission wavelength was shifted toward a longer wavelength side even relative to ν-coelenterazine.

SEQUENCE LISTING FREE TEXT

-   [SEQ ID NO: 1]

This is the nucleotide sequence of native apoaequorin.

-   [SEQ ID NO: 2]

This is the amino acid sequence of native apoaequorin.

-   [SEQ ID NO: 3]

This is the nucleotide sequence of native apoclytin-I.

-   [SEQ ID NO: 4]

This is the amino acid sequence of native apoclytin-I.

-   [SEQ ID NO: 5]

This is the nucleotide sequence of native apoclytin-II.

-   [SEQ ID NO: 6]

This is the amino acid sequence of native apoclytin-II.

-   [SEQ ID NO: 7]

This is the nucleotide sequence of native apomitrocomin.

-   [SEQ ID NO: 8]

This is the amino acid sequence of native apomitrocomin.

-   [SEQ ID NO: 9]

This is the nucleotide sequence of native apobelin.

-   [SEQ ID NO: 10]

This is the amino acid sequence of native apobelin.

-   [SEQ ID NO: 11]

This is the nucleotide sequence of native apobervoin.

-   [SEQ ID NO: 12]

This is the amino acid sequence of native apobervoin.

-   [SEQ ID NO: 13]

This is the nucleotide sequence of Renilla luciferase.

-   [SEQ ID NO: 14]

This is the amino acid sequence of Renilla luciferase.

-   [SEQ ID NO: 15]

This is the nucleotide sequence of Oplophorus luciferase.

-   [SEQ ID NO: 16]

This is the amino acid sequence of Oplophorus luciferase.

-   [SEQ ID NO: 17]

This is the nucleotide sequence of Gaussia luciferase.

-   [SEQ ID NO: 18]

This is the amino acid sequence of Gaussia luciferase.

-   [SEQ ID NO: 19]

This is the nucleotide sequence of Renilla luciferase mutant.

-   [SEQ ID NO: 20]

This is the amino acid sequence of Renilla luciferase mutant.

-   [SEQ ID NO: 21]

This is the nucleotide sequence of PCR primer RL-17N/SacI.

-   [SEQ ID NO: 22]

This is the nucleotide sequence of PCR primer RL-18C/XhoI 

1-13. (canceled)
 14. The method for producing a compound of formula (VIII) by: (1) reacting 2 amino-3,5-dibromo-6-chloropyrazine with R¹MgX (wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterocyclic group, and X is a halogen) and ZnCl₂ in the presence of a palladium catalyst to give a compound represented by general formula (V):

(wherein R¹ is the same as defined above), (2) reacting the compound represented by general formula (V) with a compound represented by general formula (VI):

(wherein R⁴ is a protecting group), in the presence of a palladium catalyst and a base to give a compound represented by general formula (VII):

(wherein R¹ and R⁴ are the same as defined above), and, (3) reacting the compound represented by general formula (VII) with tributyl(vinyl)tin in the presence of a palladium catalyst.
 15. A compound represented by formula (VIII):

wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterocyclic group, and R⁴ is a protecting group.
 16. The compound according to claim 15, wherein R¹ in formula (VIII) is hydrogen, a halogen, a substituted or unsubstituted aryl, a substituted or unsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl, an unsubstituted alkyl, an alkyl substituted with an alicyclic group, an unsubstituted alkenyl, an alkenyl substituted with an alicyclic group, an alicyclic group, a heterocyclic group, an unsubstituted alkynyl, or an alkynyl substituted with an alicyclic group.
 17. The compound according to claim 15, wherein R⁴ in formula (VIII) is methyl, methoxymethyl, tetrahydropyranyl, benzyl, 4-methoxybenzyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl, tert-butyldiphenylsilyl, or triisopropylsilyl.
 18. The compound according to claim 15 represented by formula:


19. A method for producing a compound represented by formula (IX):

wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterocyclic group, and R⁴ is a protecting group, which comprises: reacting a compound represented by formula (VIII):

wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterocyclic group, and R⁴ is a protecting group, with a methyltriphenylphosphonium salt in the presence of a base to give the compound represented by formula (IX).
 20. The method according to claim 19, wherein R¹ in formula (VIII) and formula (IX) is independently hydrogen, a halogen, a substituted or unsubstituted aryl, a substituted or unsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl, an unsubstituted alkyl, an alkyl substituted with an alicyclic group, an unsubstituted alkenyl, an alkenyl substituted with an alicyclic group, an alicyclic group, a heterocyclic group, an unsubstituted alkynyl, or an alkynyl substituted with an alicyclic group.
 21. The method according to claim 19, wherein R⁴ in formula (VIII) and formula (IX) is independently methyl, methoxymethyl, tetrahydropyranyl, benzyl, 4-methoxybenzyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl, tert-butyldiphenylsilyl, or triisopropylsilyl.
 22. The method according to claim 19, wherein the compound of formula (IX) is 2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine represented by formula:

wherein the compound of formula (VIII) is 2-(5-amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde represented by formula:

and wherein said 2-(5-amino-6-benzyl-3-vinylpyrazin-2-yl)-5-methoxybenzaldehyde is reacted with a methyltriphenylphosphonium salt in the presence of a base to give 2-amino-3-benzyl-5-(4-methoxy-2-vinylphenyl)-6-vinylpyrazine.
 23. The method according to claim 19, wherein said base is at least one base selected from the group consisting of n-butyl lithium, potassium tert-butoxide, sodium methoxide, sodium ethoxide, and lithium diisopropylamide.
 24. The method according to claim 19, wherein at least one solvent selected from the group consisting of tetrahydrofuran, diethyl ether, cyclopropyl methyl ether, tert-butyl methyl ether, dioxane, and toluene is used.
 25. The method according to claim 19, wherein the reaction temperature is 0° C. to 40° C. and the reaction time is 1 hour to 4 hours.
 26. A compound represented by formula (IX):

wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted hydrocarbon group, or a substituted or unsubstituted heterocyclic group, and R⁴ is a protecting group.
 27. The compound according to claim 26, wherein R¹ is hydrogen, a halogen, a substituted or unsubstituted aryl, a substituted or unsubstituted arylalkyl, a substituted or unsubstituted arylalkenyl, an unsubstituted alkyl, an alkyl substituted with an alicyclic group, an unsubstituted alkenyl, an alkenyl substituted with an alicyclic group, an alicyclic group, a heterocyclic group, an alkynyl, or an alkynyl substituted with an alicyclic group, in formula (IX).
 28. The compound according to claim 26, wherein R⁴ is methyl, methoxymethyl, tetrahydropyranyl, benzyl, 4-methoxybenzyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl, tert-butyldiphenylsilyl, or triisopropylsilyl, in formula (IX).
 29. The compound according to claim 26 represented by formula: 